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1、硕士学位论文HIV-1 Tat38-61(51N/55N)碱性区突变体库的构建及亲和筛选Construction of HIV-1 Tat38-61(51N/55N) Basic Region Mutation Libraries and Affinity Screening独创性声明 本人声明所呈交的学位论文是我个人在导师指导下进行的研究工作。除了文中特别加以标注和致谢的地方外，论文中不包含其它人已经发表或撰写过的研究成果。与我一同工作的同志对本研究所做的任何贡献均已在文中作了明确的说明并表示谢意。本人承担本声明的法律责任。学位论文作者签名： 签字日期： 年 月 日学位论文使用授权书声明 本
2、人完全了解第二军医大学有关保留、使用学位论文的规定，有权保留并向国家有关部门或机构送交论文的复印件和磁盘，允许论文被查阅和借阅。本人授权第二军医大学可以将学位论文的全部或部分内容编入有关数据库进行检索，可以采用影印、缩印或扫描等复制手段保存、汇编学位论文。（保密的学位论文在解密后适用本授权书）学位论文作者签名： 导师签名： 签字日期： 年 月 日 签字日期： 年 月 日第二军医大学硕士学位论文 目 录目 录中文摘要1ABSTRACT5缩略词表10前 言11第一部分 HIV-1 TAT(38-61)融合蛋白的表达及免疫原性分析131.实验材料152.实验方法203.实验结果234.讨 论27第二
3、部分 HIV-1 TAT38-61(51N/55N)碱性区突变体库的构建291.实验材料312.实验方法333.实验结果364.讨 论40第三部分 HIV-1 TAT38-61(51N/55N)碱性区突变体库的亲和筛选421.实验材料432.实验方法433.实验结果454.讨 论47总 结49综 述50参考文献55附 录 个人简历57致 谢58-57-第二军医大学硕士学位论文 中文摘要中文摘要人类免疫缺陷病毒（Human immunodeficiency virus，HIV）归属于逆转录病毒科中慢病毒属，主要经血液、性接触及母婴垂直途径等传播，可引起获得性免疫缺陷综合症（Acquired Im
4、mune Deficiency Syndrome，AIDS），即艾滋病。目前，艾滋病感染已经成为全球所面临的重大公共卫生问题。研制有效的HIV 疫苗是艾滋病防治的重要手段之一。因此，探索HIV疫苗研究的新策略和寻找新的疫苗靶点对疫苗的成功研发显得尤为重要。HIV-1反式激活蛋白(Trans-activator of transcription，Tat) 是HIV感染早期产生的一种重要调控蛋白，在HIV-1的复制、扩散和致病中起重要作用。HIV-1感染靶细胞后，胞质中合成的Tat转移到核内，与多种转录调控因子结合组成转录起始复合体（transcription initiation complex
5、, TIC）,促进病毒RNA的转录、延伸和病毒复制。此外，Tat还可被感染细胞通过多种方式分泌到胞外发挥“病毒毒素”的作用：通过诱导CD4+ T细胞、NK细胞或B细胞的凋亡，发挥其免疫抑制的作用；通过JAK/STAT信号转导途经激活HHV-8的复制，或通过RGD(Arg-Gly-Asp)与内皮细胞v3和51整合素结合，与可溶性炎症因子和血管生成因子协同促进卡波济肉瘤（Kaposis sarcoma,KS）的形成；通过影响神经细胞钙流，诱导巨噬细胞和小胶质细胞表达TGF-、TNF等神经毒性物质，发挥神经毒作用，促进艾滋病脑病的发生。Tat 碱性氨基酸富集区 (49-57aa)是Tat的穿膜和核定
6、位功能基序，是Tat蛋白的主要中和表位之一。其中和抗体可消除Tat分子所特有的穿入其他细胞发挥毒性的核心功能，从机制上能对阻止艾滋病和免疫抑制的发生有重要作用。此外，Tat碱性区序列在各亚型间高度保守，有利于产生交叉反应谱更广的抗体。本研究拟利用噬菌体展示技术分子进化平台，对天然Tat碱性区进行重组改造和筛选，构建由碱性区突变片段经随机连接肽相互连接的重复串联的噬菌体展示文库，用含有高中和活性的抗-Tat兔抗血清进行进化筛选，获得新型免疫原的代表性序列。本研究分以下三部分进行：一、 HIV-1 Tat(38-61)融合蛋白的表达及免疫原性分析功能研究表明：Tat蛋白碱性区（49-57aa）介导
7、Tat蛋白穿膜，与Tat细胞外活性密切相关。结构研究表明：Tat分子为天然非折叠蛋白，属于无规则卷曲类型的分子，缺乏稳定的二级结构和高级结构，其分子构想极不稳定，出于快速动态的变化之中，导致以Tat作为抗原，刺激效果差，高亲和力的抗体较少且容易被降解。此外，Tat碱性区只有9个氨基酸（49-57aa），为了不破坏其中和表位，我们保留了Tat核心区（38-48aa），将Tat的38-61碱性区片段构建到原核表达载体，纯化出Tat(38-61)融合蛋白并检测其免疫原性，以分析此肽段重要的中和表位是否能够得到保留。根据大肠杆菌偏好密码子优化后的HIV-1型株Tat DNA序列设计引物，用PCR方法合
8、成HIV-1 tat(38-61)序列，T/A克隆法将其克隆到pMD18-T载体进行测序。将测序正确的tat(38-61)克隆至原核表达载体pET32a，构建其原核表达质粒pET32a-tat(38-61)。将表达质粒转入E.coli BL21(DE3)中，IPTG诱导表达出相对分子量为21300的融合蛋白，并用Ni-NTA亲和层析法纯化目的蛋白。用本实验室独立制备的兔抗PEPTIDE-Tat(1-101) 血清和上海市公共卫生中心提供的HIV阳性血清进行ELISA检测，分析Tat（38-61）融合蛋白的免疫原性。结果显示：为排除融合蛋白中PET32a分子的干扰，我们选用兔抗Tat(1-101
9、)-PEPTIDE血清鉴定pET32a载体上表达的Tat(38-61)（pET32a载体与pPETTIDE 载体序列无同源性）。结果表明二者呈特异反应且反应强度低于PET32a-Tat(1-101)，与预期相符。为了彻底排除载体分子PET32a的干扰，我们用HIV阳性血清（含抗Tat单抗，无载体分子干扰）进行鉴定，结果显示表明Tat(38-61)、全长Tat二者与HIV阳性血清有基本一致的特异性反应趋势。以上ELISA测定结果说明Tat(38-61) 较好地保留了Tat碱性区的中和表位。综上所述，我们成功构建并表达出Tat(38-61)融合蛋白，碱性区肽段的重要表位得到保留，为进一步针对碱性区
10、中和抗体的制备和噬菌体展示文库的筛选奠定基础。二、 HIV-1 tat38-61(51N/55N)碱性区突变体库的构建研究表明，碱性区两个重要的位点51K和55R定点突变后，不仅其中和表位未被破坏，且Tat穿膜活性和胞外毒性消失或是被大大减弱。此外，构建适合的大容量生物大分子变异体文库是进行体外分子进化研究的关键。因此，为了扩大碱性区基因序列中被筛选的范围，我们采用含随机核苷酸序列的引物，通过Overlap PCR的方法对碱性区两个位点51K和55R进行了随机突变，使之转变成51N和55N。同时在碱性区61位后引入6个随机连接肽(SNN)6，不仅扩大文库的变异体，而且随机连接肽可与中和表位序列
11、之间相互作用,有助于产生稳定构象的新型免疫原。经过四轮PCR得到tat38-61(51N/55N)突变体片段，通过Xba I酶切后，克隆至噬菌体展示载体pCANTAB5S-LD3上，转化超级感受态Top10细胞，建立HIV-1 株Tat38-61(51N/55N)碱性区突变体文库。辅助噬菌体M13K07拯救后，得到Tat38-61(51N/55N)碱性区重组噬菌体原代展示文库。所得到的噬菌体展示文库的库容量为5.0106，滴度为2.651012 TU/ml，片段插入率为70%；14个经PCR验证的阳性克隆序列分析显示Tat38-61(51N/55N)碱性区片段中51N-55N-(SNN)6中随
12、机化的核苷酸与氨基酸均呈随机性分布。综上所述，我们成功构建了HIV-1 株Tat38-61(51N/55N)碱性区突变体文库，并且其库容、多样性、随机性均达到文库构建要求。三、 HIV-1 tat38-61(51N/55N)碱性区突变体库的亲和筛选通过第一部分的鉴定，我们发现碱性区肽段可以较好的保留其中和表位，我们用两种兔抗血清：兔抗PET32 -Tat(38-101)和兔抗PET32 -Tat(1-101)，同时对HIV-1 株Tat38-61(51N/55N)碱性区突变体文库进行亲和筛选，以期获得亲和力更强，穿膜活性更低新型免疫原的代表性序列。通过两种兔抗血清同时对文库进行两轮筛选，每轮结
13、束后，挑取单克隆进行PCR鉴定，并对PCR鉴定过的阳性克隆进行序列分析。PCR鉴定结果显示:兔抗PET32 - Tat(1-101)血清对文库筛选后，包含2个碱性区片段的单克隆在文库中所占的比例由筛选前的56%下降到4%，而包含单个碱性区片段的单克隆在文库中所占的比例由筛选前的39%上升到91%。与此结果相类似，兔抗PET32 - Tat(38-101)血清对文库筛选后，包含2个碱性区片段的单克隆在文库中所占的比例由筛选前的56%下降到0，而包含单个碱性区片段的单克隆在文库中所占的比例由筛选前的39%上升到87%。理论上，通过连接肽串联重复多个碱性区片段形成的大分子多肽，由于亲和力的关系，抗原
14、表位重复次数多具有选择性优势，容易被进化筛选获得。但两轮筛选后片段结构的变化，似乎说明碱性区中51N-55N-(SNN)6较大分子多肽对中和反应具有更重要的作用。序列鉴定分析显示:PET32 - Tat(1-101)血清对文库两轮筛选后的单克隆中，碱性区片段与载体反向连接的比例达到50%，兔抗PET32 - Tat(38-101)血清对文库两轮筛选后的单克隆中，碱性区片段与载体反向连接的比例只有6%，说明兔抗PET32 - Tat(38-101)血清对Tat(38-61)肽段的亲和力要高于PET32 - Tat(1-101)血清，这也与理论相符，因为PET32 - Tat(1-101)血清中除
15、了包含针对碱性区的抗体，还包含了针对N-端区(1-22)的抗体，而PET32 - Tat(38-101)血清只包含了针对碱性区的抗体。兔抗PET32 - Tat(1-101)血清第二轮筛选后的正确连接单克隆中，51N和55N有更多比例的脯氨酸（P）。而兔抗PET32 - Tat(38-101)血清第三轮筛选后的正确连接单克隆中，筛选到两种序列，51N-55N-(SNN)6分别是为P-D- MNKNE*(*为终止密码子)和Q-S-*KRKMN(*为终止密码子)。综上所述，我们成功用两种兔抗血清对文库进行了筛选，并用亲和力更强的兔抗PET32 - Tat(38-101) 血清进行三轮筛选后获得两种
16、51N-55N-(SNN)6代表性序列。本课题构建了pET32a-tat(38-61)原核表达质粒并纯化鉴定了Tat(38-61)融合蛋白的免疫原性,为后续使用兔抗Tat血清对碱性区突变体文库筛选奠定了基础；成功构建了HIV-1 株Tat38-61(51N/55N)碱性区突变体文库并用两种兔抗血清PET32 - Tat(38-101) 和PET32 - Tat(1-101)对文库进行了筛选，通过亲和力更强的兔抗PET32 - Tat(38-101) 血清对文库第三轮筛选后，获得了51N-55N-(SNN)6两种代表性序列P-D- MNKNE*(*为终止密码子)和Q-S-*KRKMN(*为终止密
17、码子)。本研究尝试应用以噬菌体展示技术为基础的分子进化手段对HIV-1 株Tat碱性区进行改造和筛选，为生物大分子的结构和功能研究以及新型疫苗研发策略提供了一新的可行途径。【关键词】HIV-1 Tat，碱性区，随机突变，噬菌体展示，亲和筛选第二军医大学硕士学位论文 英文摘要ABSTRACTHuman immunodeficiency virus (HIV) is attributed to the slow Retroviridae virus, spreading mainly through blood, sexual contact or mother to child such as
18、vertical channels, which can cause Acquired Immune Deficiency Syndrome (AIDS). At present, HIV infection has become a major public health problem which has to be faced by the world. Developing an effective HIV vaccine is an important means of prevention and control of AIDS. Therefore, it is very imp
19、ortant for exploring new strategies and finding new vaccine targets on the success of the vaccine research and development.HIV-1 Tat (trans-activator of transcription) is a key viral regulatory protein emerged in the early stage of HIV infection, and is necessary for viral replication, spreading and
20、 pathogenesis. After infecting target cells, the Tat generated in cytoplasm can reach the nucleus and bind with some Transcription factors to form the transcription initiation complex (TIC), which can promote transcription, extension and replication of the viral RNA. In addition, Tat can also be sec
21、reted from infected cells into the extracellular with a variety of ways in order to play a virus toxins role:playing an immunosuppressive function By inducing CD4 + T cells, NK cells or apoptosis B cells; promoting the formation of Kaposis sarcoma (KS) through some ways ,such as activating HHV-8 rep
22、lication via JAK / STAT signal transduction, or binding the RGD (Arg-Gly-Asp) with v3 and 51 integrins of the endothelial cells; promoting the incidence of HIV encephalopathy through affecting calcium flow in the nerves cells or inducing the expression of TGF-, TNF and other neurological toxic subst
23、ances. Tat basic amino acid-rich region (49-57aa) is the base sequence of functioning the trans-membrane and nuclear localization, and is also one of the main neutralization epitope. Its neutralizing antibody can eliminate the penetration of other cells, which can prevent the occurrence of AIDS and
24、immune suppression. In addition, Tat basic region sequences highly conserved among the various subtypes, which is conducive to creating a broader cross-reactivity antibodies. In This study we construct and screen of Tat basic region using phage display technology platform, and build the mutant pepti
25、de fragment library connected by random Linkers, and then screen the library using anti-Tat rabbit serum in order to obtain the original sequence of novel immunogen.1. Expression and immunogenicity analysis of HIV-1 Tat(38-61) fusion proteinsFunctional studies have shown that Tat protein basic regio
26、n (49-57aa) is playing an important role for trans-membrane function and extracellular activities mediated by Tat. Structural studies have shown that: Tat is a natural non-folded protein, and its concept is extremely unstable, so its not an ideal antigen. In addition, Tat basic region has only nine
27、amino acids (49-57aa), in order not to destroy the epitope we have maintained the Tat core region 38-48aa. The Tat basic region of 38-61 fragment was built into the prokaryotic expression vector, then we purified Tat (38-61) fusion protein and tested their immunogenicity in order to analyze whether
28、this important epitope is retained.The primers were designed according to the Tat sequence of HIV-1 strain and the preferred codons of E.coli. The tat(38-61) were synthesized in vitro by PCR and sequenced after inserted into pMD18-T vector by T/A cloning. Then the correct tat(38-61) was inserted int
29、o pET32a vector to construct prokaryotic expression plasmids pET32a-tat(38-61) and . The recombinant plasmid was transformed into E.coli BL21(DE3) for expression .The fusion protein Tat(38-61) was expressed with relative molecular weight(MW) 21300 under induction of IPTG, purified by Ni-NTA column a
30、nd testified with Two different anti-Tat rabbit serum and HIV-positive serum by ELISA. The results showed that: To exclude the interference of the carrier protein( PET32a), we identified Tat (38-61) expressed by the pET32a vector using rabbit anti-PEPTIDE-Tat (1-101) serum (the two vector sequence a
31、re no homology). The result shows that there is a specific reaction and the reactivity much lower than the native Tat, which is in line with expectations. To completely exclude the interference of the carrier protein ( PET32a), we identified Tat (38-61) using HIV-positive serum (containing only anti
32、-Tat antibody). The result shows that both Tat (38-61) and the native Tat(1-101) have the same specific reaction trend. ELISA detection of the above results suggest that the neutralization epitope of Tat basic region has been preserved by Tat (38-61).To conclude, we successfully constructed and expr
33、essed Tat(38-61) fusion proteins, and its neutralization epitope of Tat basic region has been preserved, which laid the foundation on getting the antibodies against the basic region and screening phage display library . 2. Construction of HIV-1 tat38-61 (51N/55N) basic region mutant libraryStudies h
34、ave shown that after two important sites（51K and 55R）of the Tat basic region are mutated, the neutralization epitop is not destroyed, and the activity of Tat trans-membrane and extracellular toxicity disappeared or were greatly reduced. In addition, construction of conformable large molecule mutatio
35、n library is pivotal for molecular evolution study in vitro. In order to extend the range of the screening region of amino acid in Tat basic region gene, the 51K and 55R were mutated randomly by overlap PCR amplification with the random nucleotide primers. At the same time we add to six random linke
36、r (SNN) 6 after the basic region 38-61, which not only expand the library of variants, but also help to generate a stable conformation of the novel immunogen.Through four rounds of PCR we got the tat38-61 (51N/55N) mutant fragments, and cloned into phage display vector pCANTAB5S-LD3 after digested b
37、y Xba I . then we constructed HIV-1 strain Tat38-61 (51N/55N) basic region mutant library .The phage displayed library was generated by M13K07 rescue. After detection we found that as much as 5.0106 clones were obtainded in the phage library and the titer was 2.651012 TU/ml. About 70% clones contain
38、ed inserted Tat basic region 38-61(51N/55N) fragments. Sequence analysis of 14 samples showed that nucleotide acids and amino acids at randomized 51N-55N-(SNN)6 sites distributed randomly In summary, we have successfully constructed HIV-1 strain Tat38-61 (51N/55N) basic region mutant library, and it
39、s storage capacity, diversity and randomization is all to meet the requirements of library construction.3. Affinity screening of HIV-1 tat38-61 (51N/55N) basic region mutant libraryThrough the first part of this study we know that the neutralization epitope of Tat basic region has been preserved. By
40、 using two kinds of rabbit anti-sera :PET32-Tat (38-101) and rabbit anti-PET32-Tat (1 -101), the phage libraries were screened and positive mutant clones were enriched. After two rounds screening we might get the he original sequence of those enriched mutant clones which were stronger affinity, lowe
41、r trans-membrane activity . Each round after the end of screening ,we identified the enriched mutant clones using PCR and sequence analysis.The results of PCR identification showed that: after two rounds screening by using rabbit anti-PET32 - Tat (1-101) serum , from pre-screening to screening ,the
42、proportion of the monoclonal containing two basic region fragments in the library is decreased from 56% to 4%, while the proportion of the monoclonal containing single basic region fragment in the library is increased from 39% to 91%; after two rounds screening by using rabbit anti-PET32 - Tat (38-1
43、01) serum , from pre-screening to screening ,the proportion of the monoclonal containing two basic region fragments in the library is decreased from 56% to 0, while the proportion of the monoclonal containing single basic region fragment in the library is increased from 39% to 87%. In theory, the mo
44、noclonal containing two basic region fragments could easily be obtained through evolutionary selection because of a selective advantage. It seems to suggest that the mutant sites 51N-55N-(SNN) 6 is playing a more important role for containing the neutralization epitope of Tat basic region of large t
45、han repeated fragments. The results of sequence analysis showed that: after two rounds screening by using rabbit anti-PET32 - Tat (1-101) serum , ratio of the reverse connecting between basic fragment and the vector is up to 50% .while the ratio is only 6% after two rounds screening by using rabbit
46、anti-PET32 - Tat (38-101) serum. It seems to suggest that anti-PET32 - Tat (38-101) serum has better affinity with Tat (38-61) than PET32 - Tat (1-101) serum, which is also consistent with the theory, as PET32 - Tat (1-101 ) serum contains two kinds of antibodies against the basic region and the N-t
47、erminal region (1-22), while PET32 - Tat (38-101) serum containing only antibodies against the basic region.the mutant sites 51N and 55N have a greater proportion of proline (P) in the correct monoclonal after two rounds of screening using Rabbit anti-PET32 - Tat (1-101) serum. After three rounds of
48、 screening using Rabbit anti-PET32 - Tat (38-101) serum we got the two kinds of amino acid sequences of the mutant sites 51N-55N-(SNN) 6 : the P-D-MNKNE * (* for the stop codon ), and Q-S-* KRKMN (* for the stop codon).In summary, the phage library has been successfully screened using two kinds of rabbit anti-serum, and we got the two kinds of sequences of the mutant sites 51N-55N-(SNN) 6 representative sequence after three rounds